Review Reports - Transcriptomic Analysis of <i>Ulva prolifera</i> in Response to Salt Stress

Round 1

Reviewer 1 Report

The manuscript has been well and carefully prepared and minor revision should be done by the authors before accepted.

1. L15, “a green macroalgae species” should be changed to “a green macroalgal species”.

2. L56, “Li et al. (2019)” should be changed to “Li et al. [13] ”.

3. L68，“To better understand it”，what does “it” mean here? The authors should give specific description.

4. L278, “Up-regulation of resistance-related gene expression plays a crucial role under salt stress” should be reorganized.

5. “C S5 S9” should give the specific meaning under the figures and tables.

Author Response

Dear reviewer,

Thank you very much for your sincerely working on our manuscript. Your suggestions and comments are constructive to improve our manuscript and helpful to our work in the future. We have revised the manuscript according to your suggestions. Following are the responses to your questions.

With best regards!

Question 1: L15, “a green macroalgae species” should be changed to “a green macroalgal species”.

Response 1: Thank you for your carefulness. We are sorry for the carelessness. In the revised manuscript, this inappropriate place was modified to “a green macroalgal species” according to your suggestion. Please see line 15, page 1.

Question 2: L56, “Li et al. (2019)” should be changed to “Li et al. [13]”.

Response 2: Thank you for your carefulness. We are sorry for the carelessness. These inappropriate places were modified followed by the journal guidelines for authors in the revised manuscript. This sentence has been changed to “Li et al. [14] found the expression of photosynthesis and glycerol metabolism key genes were up-regulated under salt stress by analyzing transcriptome data of Dunaliella salina” in the revised manuscript. Please see line 56-59, page 2.

Question 3: L68，“To better understand it”，what does “it” mean here? The authors should give specific description.

Response 3: Thank you for your fine suggestion. We are sorry for the incomplete information. In this place, “it” means “the molecular mechanisms for adaptation to a hypersaline environment”. The information was added in the revised manuscript. Please see line 68-69, page 2.

Question 4: L278, “Up-regulation of resistance-related gene expression plays a crucial role under salt stress” should be reorganized.

Response 4: Thank you for your fine suggestion. We have reorganized the inappropriate sentence “Up-regulation of resistance-related gene expression plays a crucial role under salt stress” to “Expression of resistance-related genes was up-regulated under salt stress” in the revised manuscript. Please see line 291, page 11.

Question 5: “C S5 S9” should give the specific meaning under the figures and tables.

Response 5: Thank you for your fine suggestion. We are sorry for the incomplete information. The “C S5 S9” means salinity 30, 50 and 90 psu treatment for 1 h, respectively. And the specific meaning of “C S5 S9” has been added in the figure and table notes in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors comprehensively analyzed the transcriptome data of Ulva prolifera at different salinities (30, 50 and 90 psu), found that stress resistance related genes up-regulated and photosynthetic oxygen release rate increased under salt stress. This manuscript provided useful information on the mechanisms underlying salinity tolerance of U. prolifera and could be interesting for the readers. However, some details are missing and minor revisions are necessary before further consideration. The specific comments are showing as follows.

Minor points:

1. Page 1 Line 15, “green tides” and “the green tide” latter are not the same. Please unite them.

2. Page 5 Line 190, Please put the logo of the figure (A and B) at the back.

3. In the discussion, read more literature and screen more stress resistance related genes, and discuss and analyze them. It would be helpful to the readability and interest of the article.

4. The format of some references should be revised. For example, Page 15, Line 420 the pages of the reference are lacking. And also pay attention to the writing of species names.

Author Response

Dear reviewer,

With best regards!

Question 1: Page 1 Line 15, “green tides” and “the green tide” latter are not the same. Please unite them.

Response 1: Thank you for your carefulness. We are sorry for the carelessness. In the revised manuscript, this inappropriate place was modified in the revised manuscript. The sentence “has caused the world's biggest green tides” has been replaced with “has caused the world's biggest green tide”. Please see line 16, page 1.

Question 2: Page 5 Line 190, Please put the logo of the figure (A and B) at the back.

Question 3: In the discussion, read more literature and screen more stress resistance related genes, and discuss and analyze them. It would be helpful to the readability and interest of the article.

Response 3: Thank you for your fine suggestion. Stress resistance-related genes are mainly about superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione reductase (GR) and so on. We have added some resistance-related genes by reading the literature. It included glutamate-cysteine ligase, tocopherol O-methyltransferase and L-ascorbate peroxidase. Please see Table 3, page 9.

Question 4: The format of some references should be revised. For example, Page 15, Line 420 the pages of the reference are lacking. And also pay attention to the writing of species names.

Response 4: Thank you for your carefulness. We are sorry for the carelessness. The inappropriate places were modified followed by the journal guidelines for authors in the revised manuscript. The species names were also checked and revised according to the journal guidelines for authors. Please see the content of the reference, pages 15-16.

Author Response File: Author Response.pdf

Reviewer 3 Report

The work seems to be complete. However, the soundness of the paper would be higher if more salt concentrations (or/end more incubation periods) were studied concerning photosynthesis research. This is especially the case of data shown in Fig 7C, where the increase and decrease of the net photosynthesis was observed, but for three points only. Moreover, the increase/decrease in gene does not strightly point to protein level increase/decrease. This should be pointed in discussion part.

Minor remarks:

ad line 147: is ms ok? it is even too short for maximal PSII determination, usuallu 800-1000ms is used; Here "the whole measutement" reffers to the fluorescence induction curve , so 300s is more reasonable?

ad line 160: was it any break between measurements? or the repetitions were one after another?

Author Response

Dear reviewer,

With best regards!

Question 1: The increase/decrease in gene does not strightly point to protein level increase/decrease. This should be pointed in discussion part.

Response 1: Thank you for your carefulness. We are sorry for the carelessness. It is true that changes in gene expression at the transcriptome level do not account for changes in expression at the protein level. Therefore, it is necessary to clarify this point in the discussion part. And the expression of protein levels has been pointed in discussion part. These sentences were “However, the up-regulation of all the resistance-related genes mentioned above was mainly at the transcriptional level, and whether they were up-regulated at the protein level remains to be explored further”. Please see line 317-320, page 12.

Question 2: ad line 147: is ms ok? It is even too short for maximal PSII determination, usually 800-1000ms is used; here "the whole measurement" refers to the fluorescence induction curve, so 300s is more reasonable.?

Response 2: Thank you for your carefulness. We are sorry for the carelessness. In the manuscript, "the whole measurement" does refer to the measurement time of the fluorescence induction curve. The whole process is about 300s. We are very sorry for the mistake in the article. This inappropriate place was modified in the revised manuscript. Please see line 150, page 4.

Question 3: ad line 160: was it any break between measurements? or the repetitions were one after another?

Response 3: Thank you for your carefulness. In our measurement process, samples were processed in batches among each treatment to minimize manual errors. Each treatment group consisted of three replicates and these samples were all independent and not duplicated for the determination. After starting the formal assay, one sample was followed by another assay.

Author Response File: Author Response.pdf

Reviewer 4 Report

In my opinion, this article can be published in this journal if the authors will improve the paper, according with the following observations:

1. I suggest authors to update the bibliography with recent studies (2022) and present the novelty elements of this paper in corellation of the recents studies added.

2. Please elaborate the section Material and Methods. Part 2.1 ad 2.2 – Please add cultivation time of microalgae. Please explain how many time the sample of macrolgae were placed in sea water with different salinity. Please explain how 1 h of treatment can influence molecular mechanisms of U. Prolifera. Please explain why other treatment times were not used.

Author Response

Dear reviewer,

With best regards!

Question 1: I suggest authors to update the bibliography with recent studies (2022) and present the novelty elements of this paper in correlation of the recent studies added.

Response 1: Thank you for your fine suggestion. We have updated and revised some of the bibliographies with recent studies (2022). For example, in bibliography 21 and 22, please see line 290-291, page 11. “When subjected to environmental stress, unfolded and misfolded proteins accumulate in the endoplasmic reticulum, thereby causing damage to the cell”, this correlate to our finding that the down-regulated genes were mainly concentrated on the metabolic pathways of protein processing in the endoplasmic reticulum. In bibliography 32, please see line 328, page 13. “Salt stress significantly increases the content of major carbohydrates, which can be involved in osmotic regulation and used as a protective agent for cell homeostasis regulation during salt stress tolerance”. In our study, the glycolytic pathway was exceptionally active in U. prolifera with salt stress (Figure 4). Carbohydrates can enter the glycolytic pathway directly, which in turn provides more ATP for coping with salt stress. Please see the content of the reference part (22; 23; 32), page 15.

[22] Feng, Y.; Ming, T.; Zhou, J.; Lu, C.; Wang, R.; Su, X. The response and survival mechanisms of Staphylococcus aureus under high salinity stress in salted foods. Foods 2022, 11, 1503.

[23] Kaur, M.; Saini, K.C.; Ojah, H.; Sahoo, R.; Gupta, K.; Kumar, A.; Bast, F. Abiotic stress in algae: Response, signaling and transgenic approaches. J Appl Phycol 2022, 34, 1843-1869.

[33] Canora, D.D.; Guasch, L.L.; Zuazo, R.S. Species-specific responses of Antarctic terrestrial microalgae to salinity stress. Comparative study in Klebsormidium sp. and Stigeoclonium sp. Czech Polar Reports 2022, 12, 89-102.

Question 2: Please elaborate the section Material and Methods. Part 2.1 ad 2.2 – Please add cultivation time of macroalgae. Please explain how many time the sample of macroalgae were placed in sea water with different salinity. Please explain how 1 h of treatment can influence molecular mechanisms of U. Prolifera. Please explain why other treatment times were not used.

Response 2: Thank you for your carefulness. We are sorry for the carelessness. The cultivation time of macroalgae have been added in the revised manuscript, which is “before the sample was used for the formal experiments, the thalli were cultured under the aforementioned conditions for 2 weeks to allow them to completely acclimatize to the environment.” Please see Part 2.1 and 2.2, page 2. All the samples of macroalgae were placed only once in sea water with different salinities.

In our study, the treatment time was set to 1h for several main considerations. On the one hand, referring to the results of some previous experiments, the transcriptome levels of plants changed significantly after 1 h of salinity treatment. Foflonker et al. (2016) reported that the green alga Picochlorum inoculated in fresh f/2 media containing 1.5 M NaCl (high salt stress) and 10 mM NaCl (low salt stress) for 1h produced significant changes in the transcriptome level. Wang et al. (2019) conducted a systematic analysis of the TcSPLs gene family including 12 members belonging to 7 groups in Tamarix chinensis, they found 1h (salt stress time) could be a critical time point of post-transcription regulation in salt stress responses. On the other hand, in this study, the salinity treatment for 1h also produced significant differences in photosynthetic physiology-related parameters (Figure 7). In addition, the upper layer of the floating thalli would be subjected to salinity stress, but not for a long time due to the action of waves. Based on these considerations, the treatment time was set as 1 h.

[1] Foflonker, F.; Ananyev, G.; Qiu, H.; Morrison, A.; Palenik, B.; Dismukes, G.C.; Bhattacharya, D. The unexpected extremophile: Tolerance to fluctuating salinity in the green alga Picochlorum. Algal Research 2016, 16, 465-472.

[2] Wang, J.; Ye, Y.; Xu, M.; Feng, L.; Xu, L.-a. Roles of the SPL gene family and miR156 in the salt stress responses of Tamarisk (Tamarix chinensis). Bmc Plant Biology 2019, 19(1).

Author Response File: Author Response.pdf